ProvisionalChapter chapter 9 Grape and Wine Metabolites: Biotechnological Approaches to Improve Wine Quality Approaches to Improve Wine Quality Fernanda Cosme, Berta Gonçalves, António Inês, Fernanda Cosme, Berta Gonçalves, António Inês, António M. Jordão and Alice Vilela António M. Jordão and Alice Vilela Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/64822 Abstract Grape metabolites can be affected by many extrinsic and intrinsic factors, such as grape variety, ripening stage, growing regions, vineyard management practices, and edaphoclimatic conditions. However, there is still much about the in vivo formation of grape metabolites that need to be investigated. The winemaking process also can create distinct wines. Nowadays, wine fermentations are driven mostly by single-strain inoculations, allowing greater control of fermentation. Pure cultures of selected yeast strains, mostly Saccharomyces cerevisiae, are added to grape must, leading to more predictable outcomes and decreasing the risk of spoilage. Besides yeasts, lactic acid bacteria also play an important role, in the final wine quality. Thus, this chapter attempts to present an overview of grape berry physiology and metabolome to provide a deep understanding of the primary and secondary metabolites accumulated in the grape berries and their potential impact in wine quality. In addition, biotechnological approaches for wine quality practiced during wine alcoholic and malolactic fermenta- tion will also be discussed. Keywords: grape physiology, grape metabolites, wine biotechnology, alcoholic fer- mentation, malolactic fermentation, microbial metabolites 1. Introduction Grape berry chemical composition is complex, containing hundreds of compounds. Water (75– 85%) is the main component followed by sugars and then organic acids. Other important compounds include amino acids, proteins, and phenolic compounds. Berry sugar composition © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, and reproduction in any medium, provided the original work is properly cited. distribution, and reproduction in any medium, provided the original work is properly cited. 188 Grape and Wine Biotechnology has a key role in wine quality, since it determines alcohol content in wines [1]. Grape sugar, acidity, pH, and color are considered to mark harvest. Bouquet and flavor are related to the winemaker’s expertise, stabilization, and storage processes, but primarily they are related to grape varietal character and its particular expression in a given terroir. Nowadays, wine fermentations are driven mostly by single-strain inoculations, allowing greater fermentation control, leading to more predictable outcomes and decreasing the risk of spoilage by other yeasts [2]. During must fermentation, Saccharomyces cerevisiae produces a plethora of active-aroma secondary metabolites and releases many aroma compounds from inactive precursors present in grape juice, which significantly affect the sensory quality of the final wine [3, 4]. Besides yeasts, lactic acid bacteria (LAB) are members of the normal microbiota that appears in all type of wines (white and red), and, therefore, they also play an important role in their final quality. Malolactic fermentation (MLF), a long-standing process of deacidi- fication in winemaking carried by LAB, is a reaction of l-malic acid decarboxylation to l-lactic acid. Complex metabolic activities also occur, thus suggesting that MLF can positively or negatively affect the final wine quality [5, 6]. 2. Grape berry physiology and metabolome 2.1. Morphology and anatomy of grape berries After successful pollination and fertilization of ovules within a flower berry development initiates [7]. The formation and growth of grape (Vitis vinifera) berries follows a double sigmoid pattern with three distinct phases [8]: I, rapid cell division and expansion in green berries; II or lag phase, in which cell expansion ceases; and III, in which growth is reinitiated and the fruit matures. The berry fruit comprises up to four seeds surrounded by the inner endocarp, the middle mesocarp, pulp or flesh, and the outer exocarp or skin [8, 9] (Figure 1). Figure 1. Structure of a ripe grape berry. Illustrated by Sílvia Afonso. The exocarp consisting of a cuticle-covered epidermis, which represents 5–18% of the fresh weight of the fruit [10] and several layers of underlying thick-walled cells of hypodermis, Grape and Wine Metabolites: Biotechnological Approaches to Improve Wine Quality 189 http://dx.doi.org/10.5772/64822 contains most of the skin flavonoids [11], notably anthocyanins in the outermost layers of the red grape varieties [8], interspersed with cells rich in needle-like crystals (raphides) [12]. Epidermis has non-photosynthetic cells with vacuoles containing large oil droplets [8]. Small berries have greater color, tannins, and flavor compounds than large berries because skin has a higher percentage of the total mass of small berries [7]. Scanning electron microscopy showed very few but functional stomata on young berries and wax-filled stomata on older berries [13], which accumulate polyphenolics and abnormally high concentrations of silicon and calcium in the peristomatal protuberances of up to 200 µm diameter [14]. At harvest, the cuticle of grape berry had an amorphous outer region and a mainly reticulate inner region [15]. During fruit development, the composition of the cuticular waxes changed, being oleanolic acid the main constituent, representing 50–80% of the total weight [16]. The soft wax was a mixture of long chain fatty acids (C16 and C18 fatty acid esters [17]), alcohols, aldehydes, esters, and hydrocarbons [18]. The mesocarp consists of thin-walled parenchyma [12]. The cells are round to ovoid and contain large vacuoles, which are the primary sites for the accumulation of sugars and phenolics [8], water, and organic acids [9] during grape berry ripening. According to Coombe [19], the translucent and hydrated mesocarp composes 85–87% of the berry’s spherical volume. Altogether these make up 99.5% of the juice mass and hence are the major determinants of berry size and quality [9, 20]. The remaining 0.5% of berry components are phenolics, terpe- noids, lipids, cellulose, and pectin [20]. The endocarp consists of crystal-containing cells (druses) and an inner epidermis [12]. Grape seeds are contained in locules (Figure 1), and are composed of an outer seed coat, the endosperm, and the embryo [9]. As with most seeds, the endosperm comprises the bulk of the grape seed and serves to nourish the embryo during early growth. The normal or perfect number of seeds in the grape is four [9], but lack of ovule fertilization or ovule abortion reduces the number of developing seeds, generally resulting in smaller berry size [7]. Based upon recent molecular evidence, auxin is synthesized in the ovule and transported to the pericarp upon fertilization, where it induces gibberellin (GA) biosynthesis. The GA then degrades DELLA proteins that repress ovary growth and fruit initiation [21]. The size of mature berries at harvest is also a function of the number of cells divisions before and after flowering, extent of growth of these cells [22], and the extent of preharvest shrinkage [23]. High level of tannins is observed in the seed coat [9, 11]. Similar to the tannins and phenols found in the flesh, these tannins also decline greatly on a per-berry basis after véraison [24]. Berry vascular tissue develops directly from that of the ovary. It consists primarily of a series of peripheral bundles that ramify throughout the outer circumference of the berry and axial bundles that extend directly up through the stem [8]. Grape berry is provided through the berry stem or pedicel by a vascular system composed of xylem and phloem vessels [25]. Water, minerals, hormones, and nutrients are transported from the root system throughout the vine by the xylem tissue [25]. Present evidence indicates that in the final stages of grape develop- ment, water movement through the xylem vessels decreases markedly [25]. But, it seems that the fruit is not hydraulically isolated from the parent grapevine by xylem occlusion then, 190 Grape and Wine Biotechnology rather, is “hydraulically buffered” by water delivered via the phloem [9]. Berry is also supplied by the phloem, which is the vasculature involved in photosynthate (sucrose) transport from the canopy to the vine [25]. 2.2. Grape primary and secondary metabolites 2.2.1. Sugars One of the main features of the grape-ripening process is the accumulation of sugars in the form of glucose and fructose within the cellular medium, specific in vacuole. In addition, sugar content is an important indicator often used to assess ripeness and to mark grape harvest. But, it is also possible to quantify small traces of sucrose in V. rotundifolia and hybrids between V. labrusca and V. vinifera grapevines [26]. Liu et al. [27] analyzed sugar concentration of 98 different grape cultivars and concluded that glucose (45.86–122.89 mg/mL) and fructose (47.64– 131.04 mg/mL) were the predominant sugars in grape berries.